Development of PCR-based markers for identification of wheat HMW glutenin Glu-1Bx and Glu-1By alleles

Background In common wheat (Triticum aestivum L.), allelic variations in the high-molecular-weight glutenin subunits Glu-B1 locus have important effects on grain end-use quality. The Glu-B1 locus consists of two tightly linked genes encoding x- and y-type subunits that exhibit highly variable frequencies. However, studies on the discriminating markers of the alleles that have been reported are limited. Here, we developed 11 agarose gel-based PCR markers for detecting Glu-1Bx and Glu-1By alleles. Results By integrating the newly developed markers with previously published PCR markers, nine Glu-1Bx locus alleles (Glu-1Bx6, Glu-1Bx7, Glu-1Bx7*, Glu-1Bx7 OE, Glu-1Bx13, Glu-1Bx14 (−) , Glu-1Bx14 (+)/Bx20, and Glu-1Bx17) and seven Glu-1By locus alleles (Glu-1By8, Glu-1By8*, Glu-1By9, Glu-1By15/By20, Glu-1By16, and Glu-1By18) were distinguished in 25 wheat cultivars. Glu-1Bx6, Glu-1Bx13, Glu-1Bx14 (+)/Bx20, Glu-1By16, and Glu-1By18 were distinguished using the newly developed PCR markers. Additionally, the Glu-1Bx13 and Glu-1Bx14 (+)/Bx20 were distinguished by insertions and deletions in their promoter regions. The Glu-1Bx6, Glu-1Bx7, Glu-1By9, Glu-1Bx14 (−), and Glu-1By15/By20 alleles were distinguished by using insertions and deletions in the gene-coding region. Glu-1By13, Glu-1By16, and Glu-1By18 were dominantly identified in the gene-coding region. We also developed a marker to distinguish between the two Glu-1Bx14 alleles. However, the Glu-1Bx14 (+) + Glu-1By15 and Glu-1Bx20 + Glu-1By20 allele combinations could not be distinguished using PCR markers. The high-molecular-weight glutenin subunits of wheat varieties were analyzed by ultra-performance liquid chromatography and sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the findings were compared with the results of PCR analysis. Conclusions Seven Glu-1Bx and four Glu-1By allele detection markers were developed to detect nine Glu-1Bx and seven Glu-1By locus alleles, respectively. Integrating previously reported markers and 11 newly developed PCR markers improves allelic identification of the Glu-B1 locus and facilitates more effective analysis of Glu-B1 alleles molecular variations, which may improve the end-use quality of wheat. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-024-05100-w.


Background
Wheat quality is mainly based on wheat gluten protein, which has a wide range of effects on dough properties, and protein content and composition are key qualitydetermining parameters [1].Gluten is a typical waterinsoluble protein polymer composed of disulfide bonds and noncovalent hydrogen bonds between polymeric glutenin and monomeric gliadins [2].Glutenin influences the strength and elasticity of wheat dough [3], whereas gliadins are responsible for its extensibility and viscosity [4].Glutenin proteins include two major subunits, highmolecular-weight glutenin subunits (HMW-GSs) and low-molecular-weight glutenin subunits (LMW-GSs), which are the major proteins affecting the end-use quality of wheat [3,5].The genes encoding the HMW-GS, namely, Glu-A1, Glu-B1, and Glu-D1, are located on the long arms of chromosomes 1A, 1B, and 1D, respectively [4,6].Each Glu-1 locus consists of two tightly linked genes, designated as the x-type and y-type subunits that are highly conserved, contain repeated domains, and exhibit multiple alleles [7][8][9].HMW-GSs account for only about 5%-10% of grain protein, but allelic variations in HMW-GSs have been reported to account for up to 50-70% of the variation in bread-making quality [10][11][12][13][14][15].Three, 11, and six alleles at the Glu-A1, Glu-B1, and Glu-D1 loci, respectively, were systematically identified in 1983 by isolating HMW-GSs using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [16].Extensive polymorphisms were detected at all three Glu-1 loci.The degree of polymorphism has continued to increase with analyses of different landraces, wild species, and wheat relatives [5,17].The Grain Genes 2.0 database identified 22 alleles for Glu-A1, 52 for Glu-B1, and 36 for Glu-D1 [18].
Identification of HMW glutenin in wheat remains a high priority in wheat-breeding programs to determine which genotypes should be used in breeding programs because of the large variations in allelic composition among varieties.Although new genotypes continue to be reported, PCR markers cannot distinguish among all genotypes.In addition, the discriminant information of previously reported markers was limited to only a few alleles.SDS-PAGE and HPLC/UPLC analyses are not only time-consuming, but in some cases size-indistinguishable, necessitating the development of PCR markers that can distinguish the genotype of a breed.In this study, we developed PCR markers to distinguish nine Glu-1Bx alleles and seven Glu-1By alleles located at the Glu-B1 locus by using several specific SNPs and indels in 25 wheat varieties.These markers could be useful for marker-assisted selection of Glu-B1 alleles in wheat quality improvement programs.

Plant materials
A total of 25 wheats were provided by the Korean Agricultural Culture Collection (KACC) (htpp://genebank.rda.go.kr).Formal identification of wheat varieties used in this study were performed by KACC.Three IT numbers (registration numbers for plants at the NARO Institute of Agrobiological Sciences National) were assigned to the Avalone cultivar.Information of HMW-GS allele compositions and IT numbers is listed in Table 1.The respective countries of origin and published and corrected Glu-B1 alleles are also indicated (Table 1).

Genomic DNA extraction
A total of 27 hexaploid wheat (Triticum aestivum L.) plants were grown in a Petri dish with moisture paper.Genomic DNA was extracted from the leaf tissue using the Higene ™ Genomic DNA Prep kit (solution type; BioFACT, Korea).The quality of the genomic DNA was assessed using a NanoDrop 1000 spectrophotometer (Thermo Scientific, MA, USA), and the integrity of the DNA was checked using 1% agarose gel electrophoresis.

Glutenin protein analysis using SDS-PAGE
Glutenin was extracted from single wheat grains using a previously reported HMW-GS extraction protocol [42,43].Protein (approximately 10 μg) extracted from grains of different varieties was separated using 10% SDS-PAGE and visualized using Coomassie Brilliant Blue R + 250 staining solution (Bio-Rad, CA, USA).

Glutenin analysis using ultra-performance liquid chromatography
Glutenin analysis was performed by crushing single grains of wheat, and the glutenin extraction method was performed as described previously [42,43].The extracted glutenin was analyzed using a UPLC system (Alliance e2695, Waters Corp., MA, USA) with an ACQUITY UPLC Peptide BEH C18 column (300A, 1.7 μm, 2.1 mm × 50 mm) and a photodiode array detector.The mobile phases were H 2 O containing 0.1% trifluoroacetic acid (A) and acetonitrile containing 0.1% trifluoroacetic acid (B).The injection volume of the dissolved samples was 3 μL and the flow rate was 0.55 μL/min.The solvent gradient was changed from 21 to 47% (B) from 0 to 30 min, and the column and sample temperatures were set to 55 °C and 10 °C, respectively.
Therefore, we developed a PCR-based marker that detects a 185 bp insertion without a 43 bp insertion in the promoter (Fig. 2e, Fig. S2).Similar to the marker reported by Xu et al. [31] (PS6, Fig. 2f ), this marker showed good detection ability for the 185 bp insertion in Glu-1Bx20 (Suwon15, Suwon28, and Suwon42).One of the Glu-1Bx7 variants, Glu-1Bx7*, is characterized by an 18 bp deletion in the repeat domain corresponding to an additional hexapeptide motif [46], and a Glu-1Bx7* detection marker using this 18 bp deletion has been reported by Espi et al. [35].The reverse primer for Glu-1Bx7* reported by Espi et al. [35] was modified in this study.However, Glu-1Bx17 also lacks an 18 bp repeat domain corresponding to an extra hexapeptide motif and was detected by PS7 (Fig. 2g).In addition to the 18 bp deletion, Glu-1Bx17 is characterized by a 108 bp deletion in the coding sequence; therefore, a marker capable of distinguishing Glu-1Bx17 with a 108 bp deletion from other Glu-1Bx genes has been reported [28,31] (PS8, Fig. 2h).With this marker, Glu-1Bx6 was detected to be 45 bp larger than other Glu-1Bx genes except for Glu-1Bx17.Additionally, we found that Gobun, Eunpa, and Anbaek contained Glu-1Bx7* instead of Glu-1Bx7 [47,48] (Fig. 2g).

Detection of the Glu-1Bx6 subunit
Glu-1Bx6 was detected as a larger PCR fragment than the other Glu-1Bx alleles in PS8.Additionally, Glu-1Bx6 was distinguished from other Glu-1Bx, except for Glu-1Bx20, by a 15 bp insertion in the coding regions [32] (PS9, Fig. 2i).However, Glu-1Bx20 also contained a 15 bp insertion in the coding region (Fig. 2i).Therefore, a dominant marker for Glu-1Bx6 was developed to detect a size of 457 bp using SNP (PS10).As shown in Fig. 2j, the PCR products from three Avalon cultivars were detected using this marker.

Detection of Glu-1Bx13 alleles
The tandem 54 bp duplication at position -400 of the promoter region contains a "cereal box", which has been implicated in seed-specific expression [49].However, the promoter region of Glu-1Bx13 does not contain a 54 bp replication [40].To discriminate of Glu-1Bx13 from other Glu-1Bx alleles in the promoter, we developed a codominant primer set to detect Glu-1Bx13 and non-Glu-1Bx13 with sizes of 365 and 419 bp, respectively (PS11, Fig. 2k).Three cultivars (Jeokjoong, Baekjoong, and Joeun) showed a 54 bp deletion in comparison with the other cultivars.In addition, we developed a PCR primer set to detect the Glu-1Bx13 coding region.This primer set dominantly detected Glu-1Bx13 with a size of 254 bp (PS12, Fig. 2l).

Detection of the Glu-1Bx20 subunit
Glu-1Bx20 contains a 185 bp insertion in the promoter region and was detected by PS2, PS5, and PS6.However, some types of Glu-1Bx14 have been reported to contain 185 bp insertion in the promoter region [40,45].Therefore, we developed a set of primers (PS13) for the detection of the Glu-1Bx20 coding region with a size of 501 bp using an SNP (Fig. 2m).This primer set specifically detected Glu-1Bx20 in three cultivars (Suwon15, Suwon28, and Suwon42).

Detection of Glu-1Bx14 subunits
Two German bread wheat cultivars, Hanno and Imbros, carry Glu-1Bx14 (−) along with the Glu-1By15 subunits [21].The Glu-1Bx14 (+) sequence was highly similar with to that of Glu-1Bx20 [31,39].However, the three variants (Troll, Hanno, and Imbros) not only did not contain insertions of 43 bp and 185 bp in the promoter region but were also specifically detected by PS1 in the detection of Glu-1Bx7 homologs (Fig. 2).Since two accession numbers were registered in NCBI, we compared the sequences corresponding to the accession numbers AY367771 and KF733216 (Fig. S3).These two accession numbers showed 94% similarity in the nucleotide sequence, and the KF733216 sequence showed an 18 bp deletion in the coding sequence in comparison with other Glu-1Bx alleles.Next, we developed a PCR marker for detection of Glu-1Bx14 (−) using the 18 bp indel (PS14, Fig. 2n).This marker differentiated Glu-1Bx14 (−) from Glu-1Bx7 homologs and Glu-1Bx17.Three cultivars (Troll, Hanno, and Imbros)  showed a 18 bp deletion.We also tested RANEE (Glu-1Bx14 + Glu-1By15), a cultivar containing Glu-1Bx14.

Detection of Glu-1By8 and Glu-1By18 subunits
Lei et al. [28] reported two markers (PS15 and PS16) that could differentiate Glu-1By8 from Glu-1Bx8* and Glu-1Bx18 alleles, which are generally difficult to distinguish using SDS-PAGE.A pair of AS-PCR primers (PS15) discriminated Glu-1By8, which produced a 527 bp fragment, while non-Glu-1By8 alleles showed negative results for PS15 (Fig. 5a).Previously, a pair of AS-PCR primers (PS16) was used to discriminate between Glu-1By18 and Glu-1By8 genes.In this study, no PCR product was detected from Glu-1By8 using this primer set; however, a PCR product of 527 bp was amplified from the other Glu-1By alleles, but the amplified Glu-1By15 and Glu-1By20 PCR products were weak (Fig. 5b).These markers could distinguish Glu-1By8 from other Glu-1By alleles, but could not distinguish Glu-1By8* and Glu-1By18 from other Glu-1By alleles (Fig. 5a, b).Therefore, in this study, we developed a set of specific primers to detect a 543 bp PCR product to distinguish Glu-1By18 from other Glu-1By alleles (PS17, Fig. 5c).With PS17, a single band of 543 bp was specifically detected in three cultivars (Joongmo2008, Suwon92, and Suwon105) containing Glu-1By18.

Detection of Glu-1By9 and Glu-1By15/20 subunits
Two specific primer sets (PS18 and PS19) for Glu-1By9 allele detection have been reported to assay a 45 bp deletion [28,37].In this study, PS18 identified a PCR product that was 45 bp smaller in Glu-1By9 than in non-Glu-1By9, and two weak bands were detected for the Glu-1By15 and Glu-1By20 alleles (Fig. 5d).PS19 also identified a PCR product 45 bp smaller in Glu-1By9 than in non-Glu-1By9.One and two larger bands were detected for Glu-1By15 and Glu-1By20 alleles, respectively (Fig. 5e).These two primer sets detected a size of approximately 900 bp, and prolonged electrophoresis was required to distinguish the indels.In addition, the nucleotide sequences of Glu-1By15 and Glu-1By20 were highly similar and could not be distinguished.Therefore, in this study, we designed two additional primer sets to distinguish between Glu-1By15 and Glu-1By20, which produced a smaller PCR product with a 45 bp deletion (PS20-PS21, Fig. 5f-g).These markers also showed a 45 bp indel in Glu-1By9, but one large-sized band (PS20-PS21) were detected for Glu-1Bx15 and Glu-1Bx20, respectively.Therefore, these primer sets (PS18-21) could distinguish Glu-1By15 and Glu-1By20 from other alleles; however, Glu-1By15 and Glu-1By20 were detected in the same pattern and could not be distinguished from each other.

Detection of the Glu-1By16 subunit
Lei et al. [24] previously reported a PCR marker (PS22) for the detection of the Glu-1By16 allele.However, this marker showed multiple bands (three bands for Glu-1Bx16, zero or one band for Glu-1Bx15 and Glu-1Bx20, and two bands for the other Glu-1By alleles) (Fig. 5h).Therefore, we developed a Glu-1By16-specific PCR-based marker with a product size of 558 bp (PS23, Fig. 5i).This primer set specifically detected the Glu-1By16 allele in three cultivars: Jeokjoong, Baekjoong, and Joeun.

Analysis of HMW-GS proteins using SDS-PAGE
HMW-GS glutenin subunits were compared using SDS-PAGE to confirm the presence of PCR markers in the wheat grains (Fig. 7).Glu-1Bx7 and Glu-1Bx7* differed by five amino acids and showed calculated molecular weights of 85.31 kDa and 84.71 kDa (www.bioin forma tics.org), respectively, and the two proteins could not be distinguished by SDS-PAGE.Similarly, the molecular weights of Glu-1Bx14 (−) , Glu-1Bx14 (+) , and Glu-1Bx20 were calculated to be 84.53kDa, 86.23 kDa, and 86.11 kDa, respectively, which were distinct from those of Glu-1Bx14 (−) and Glu-1Bx20.However, Glu-1Bx14 (+) and Glu-1Bx20 could not be distinguished by SDS-PAGE.Additionally, the molecular weights of Glu-1By8 and Glu-1By18 were calculated to be 77.38 kDa and 77.44 kDa, respectively.Glu-1By8 and Glu-1By8* proteins were highly identical in size and could not be distinguished by SDS-PAGE.The molecular weights of Glu-1By15 and Glu-1By20 were calculated to be 77.40 kDa and 77.43 kDa, respectively, and were also not distinguishable from each other.

Discussion
Allele variations in HMW-GSs are highly related to wheat baking quality, and among the three Glu-1 loci, Glu-B1 shows the greatest allele variations in both tetraploid and hexaploid wheat [50].Therefore, various mass spectrometry techniques, SDS-PAGE analyses, and molecular markers have been developed to identify HMW-GSs.However, new genotypes continue to be reported, and there are few genotypes that can be distinguished using the developed markers.In addition, many previously reported PCR-based markers often performed simple relative comparison analyses without comparing various alleles.Moreover, the primer sequences and accession numbers were incorrect in some reports [35,39].In    addition, in some cases, in genotypes of alleles that could not be distinguished by SDS-PAGE and LC analyses were re-evaluated [43,47].
In this study, 11 novel Glu-B1 allele identification markers were developed, and together with previously reported markers, they could be used to distinguish nine Glu-1Bx alleles (Glu-1Bx6, Glu-1Bx7, Glu-1Bx7*, Glu-1Bx7 OE , Glu-1Bx13, Glu-1Bx14 (−) , Glu-1Bx17, and Glu-1Bx14 (+) /20) and seven Glu-1By alleles (Glu-1By8, Glu-1By8*, Glu-1By9, Glu-1Bx16, Glu-1By18, and Glu-1By15/20).These findings confirmed that the Glu-1By allele of Avalone was Glu-1By8*, not Glu-1By8, and the Glu-1Bx allele of the three cultivars, Gobun, Eunpa, and Anbaek, was confirmed to be Glu-1Bx7*, not Glu-1By7.Geng et al. (2014) reported three Chinese and 11 European cultivars among 505 Chinese and 160 European cultivars with a 43 bp insertion.Cultivars containing 43 bp insertion were rare and had a high proportion of the Glu-1Bx7 gene.Among them, Glu-1Bx6 containing 43 bp insertion was identified in European cultivars 'GK Bence' and 'Komorowska-pol' , and Glu-1Bx14 containing 43 bp insertion was found in European cultivars 'Funo' and ' Amarelo de barba branca' [40].The present study showed that the Glu-1Bx6 allele from Avalon and the Glu-1Bx14 allele from Troll, Hanno, and Imbros are cultivars that do not contain 43 bp insertion in the promoter.Therefore, two types of promoters possibly exist in the Glu-1Bx6 and Glu-1Bx14 alleles.Cases with and without 43 bp insertions were identified; however, Glu-1Bx6 and Glu-1Bx14 with a 43 bp insertion are rare.Additionally, two Glu-1Bx14 allele accession numbers have been registered with NCBI and were distinguishable by 18 bp indels.Three cultivars-Hanno, Imbros, and Troll, showed a 18 bp deletion in Glu-1Bx14 in comparison with the other Glu-1Bx alleles, but RNAEE did not, allowing the distinction of these cultivars using the markers we developed.
In this study, two DNA polymerases were used for amplification.Unlike the conditions described in the previous studies, the annealing temperature for the optimal conditions differed depending on the DNA polymerase.Therefore, when analyzing markers, the annealing temperature must be set according to the DNA polymerase and equipment.
Glu-1Bx14 (+) and Glu-1By20, and Glu-1By15 and Glu-1By20 could not be distinguished using UPLC analysis.Additionally, it was not easy to distinguish the sizes of the two proteins in SDS-PAGE analysis.These findings highlight the need to develop PCR-based markers that can easily distinguish between these two allele combinations.However, we were could not distinguish Glu-1Bx14 (+) from Glu-1Bx20, nor could it distinguish Glu-1By15 from Glu-1By20 with the PCRbased marker.In addition, since many alleles were not tested in this study and more alleles may occur, the primers developed here may not be fully applicable.However, the most commonly used allele combinations can be distinguished by PCR-based markers developed in this study.Additionally, these results suggesting that the Glu-A1 and Glu-D1 alleles also need to be reassessed through PCR-based markers.

Conclusions
HMW-GS allele composition is a crucial factor in determining end-use quality, and allele identification is an essential task in wheat breeding programs.Seven Glu-1Bx and four Glu-1By allele detection markers were developed to detect nine Glu-1Bx and seven Glu-1By locus alleles, respectively.The discrimination of Glu-B1 locus alleles can be improved and the most commonly used allele combinations can be identified by integrating previously reported markers and 11 newly developed PCR markers.However, these PCR markers cannot distinguish the Glu-1Bx14 (+) + Glu-1By15 and Glu-1Bx20 + Glu-1By20 combination; therefore, further research is needed.The developed markers can facilitate more effective analysis of molecular variations in the Glu-B1 allele, thereby improving the end-use quality of common wheat.

Fig. 5
Fig. 5 PCR analysis of Glu-1By alleles in wheat varieties.The numbers above the figure are the same as the variety numbers in Table 1.The primer set numbers in the figure are the same as in the Table 3. DM, DNA size marker; PS, primer set.Arrows indicate indels

Fig. 6
Fig. 6 Identification of HMW-GS Glu-1Bx and Glu-1By in 24 wheat cultivars by ultra-performance liquid chromatography.Glu-1Bx and Glu-1By alleles are shown in blue.AU, arbitrary units; RT, retention time

Table 1
List of wheat varieties used in this study.The respective countries of origin and published and corrected Glu-B1 alleles are indicated a IT

Table 2
Primers used in this study for detection of Glu-1Bx alleles PS Primer set, CDS Coding sequence, AT Annealing temperature, new Newly developed marker

Table 1
. The primer set numbers in the figure are the same as in Table 2. DM, DNA size marker; PS, primer set.Arrows indicate indels

Table 1 .
The primer set numbers in the figure are the same as in the Table3.DM, DNA size marker; PS, primer set.Arrows indicate indels

Table 3
Primers used in this study for detection of Glu-1By alleles PS Primer set, CDS Coding sequence, ND Not determined, AT Annealing temperature, new newly developed marker